US9012227B2 - ω-Aminocarboxylic acids, ω-aminocarboxylic acid esters, or recombinant cells which produce lactams thereof - Google Patents

ω-Aminocarboxylic acids, ω-aminocarboxylic acid esters, or recombinant cells which produce lactams thereof Download PDF

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US9012227B2
US9012227B2 US12/742,318 US74231808A US9012227B2 US 9012227 B2 US9012227 B2 US 9012227B2 US 74231808 A US74231808 A US 74231808A US 9012227 B2 US9012227 B2 US 9012227B2
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enzyme
aminocarboxylic acid
aminocarboxylic
cell
acid
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US20100324257A1 (en
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Andreas Karau
Volker Sieber
Thomas Haas
Harald Haeger
Katrin Grammann
Bruno Buehler
Lars Blank
Andreas Schmid
Guido Jach
Bernd Lalla
Andreas Mueller
Katrin Schullehner
Peter Welters
Thorsten Eggert
Andrea Weckbecker
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Evonik Operations GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/14—Lactams
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids

Definitions

  • the present invention relates to cells that are genetically modified relative to their wild type, a method for the production of a genetically modified cell, the cells obtainable by this method, a method for the production of ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or of lactams derived from ⁇ -aminocarboxylic acids, the ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids obtainable by this method, a method for the production of polyamides based on ⁇ -aminocarboxylic acids or on lactams and the polyamides obtainable by this method.
  • Polyamides are polymers whose repeating units (monomers) possess the amide group as a characteristic feature.
  • polyamide 6 a product that is widely used in industry, is obtained by ring opening polymerization of ⁇ -caprolactam
  • polyamide 12 which is also industrially important, is obtained by ring opening polymerization of laurinlactam.
  • Copolymers of lactams, such as copolymers of ⁇ -caprolactam and laurinlactam (“polyamide 6/12”) are also of considerable commercial importance.
  • ⁇ -caprolactam is usually carried out by reacting cyclohexanone with the hydrogensulphate or the hydrochloride of hydroxylamine with formation of cyclohexanone oxime. This is converted by a Beckmann rearrangement into ⁇ -caprolactam, often with the use of concentrated sulphuric acid as catalyst. Cyclohexanone is usually produced by catalytic oxidation of cyclohexane with oxygen of the air, cyclohexane being obtained in its turn by hydrogenation of benzene.
  • laurinlactam is particularly expensive. On an industrial scale this first involves the trimerization of butadiene, with formation of cyclododecatriene. The cyclododecatriene is then hydrogenated with formation of cyclododecane and the cyclododecane obtained is oxidized with formation of cyclododecanone. The cyclododecanone thus obtained is then reacted with hydroxylamine to cyclododecane oxime, which is then converted in a Beckmann rearrangement to laurinlactam.
  • EP-A-0 748 797 describes a method for the production of lactams from dinitriles, in which the dinitrile is hydrogenated to aminonitrile and the aminonitrile is converted by cyclizing hydrolysis to the lactam.
  • the present invention was based on the aim of overcoming the disadvantages arising from the prior art.
  • the present invention was based on the aim of providing a method by which lactams, in particular laurinlactam, can be formed in the fewest possible steps and with formation of the minimum possible amount of by-products.
  • Another aim of the present invention was to provide a method by which lactams, in particular laurinlactam, can be produced from renewable raw materials.
  • a contribution to achievement of the aforementioned aims is provided by a cell, which has been genetically modified relative to its wild type so that, in comparison with its wild type, it is able to produce more ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or more lactams derived from ⁇ -aminocarboxylic acids, starting from carboxylic acids or carboxylic acid esters.
  • Such a cell can be used in order to produce ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids by fermentation from carboxylic acids or carboxylic acid esters, for example from lauric acid or lauric acid esters.
  • the formulation “that in comparison with its wild type it is able to produce more ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or more lactams derived from ⁇ -aminocarboxylic acids, starting from carboxylic acids or carboxylic acid esters” also applies to the case when the wild type of the genetically modified cell is not able to form any ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or any lactams derived from ⁇ -aminocarboxylic acids, or at least no detectable amounts of these compounds and it is only after the genetic modification that detectable amounts of these components can be formed.
  • a “wild type” of a cell preferably denotes a cell whose genome is in a state such as arose naturally by evolution. The term is used both for the whole cell and for individual genes. The term “wild type” therefore in particular does not include such cells or such genes whose gene sequences have been altered at least partially by man by recombinant methods.
  • the genetically modified cell prefferably has been genetically modified so that in a defined time interval, preferably within 2 hours, still more preferably within 8 hours and most preferably within 24 hours, it forms at least twice, especially preferably at least 10 times, even more preferably at least 100 times, and yet more preferably at least 1000 times and most preferably at least 10000 times more ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids than the wild-type cell.
  • the increase in product formation can be determined for example by cultivating the cell according to the invention and the wild-type cell each separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specified time interval in a suitable nutrient medium and then determining the amount of target product ( ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids) in the nutrient medium.
  • target product ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids
  • the cells according to the invention can be prokaryotes or eukaryotes. They can be mammalian cells (such as human cells), plant cells or microorganisms such as yeasts, fungi or bacteria, microorganisms being especially preferred and bacteria and yeasts being most preferred.
  • Suitable bacteria, yeasts or fungi are in particular those bacteria, yeasts or fungi that have been deposited in the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, abbreviated to DSMZ), Brunswick, Germany, as strains of bacteria, yeasts or fungi.
  • Cells that are especially preferred according to the invention are derived from cells of the genera Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia and Clostridium , with Escherichia coli, Corynebacterium glutamicum and Pseudomonas putida being especially preferred and Escherichia coli being most preferred.
  • the latter displays, in comparison with its wild type, increased activity of at least one of the following enzymes:
  • the term “increased activity of an enzyme”, as used above in connection with the enzyme E I and hereinafter in connection with the enzymes E II etc., is preferably to be understood as increased intracellular activity.
  • an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences that code for the enzyme, using a strong promoter or employing a gene or allele that codes for a corresponding enzyme with increased activity and optionally by combining these measures.
  • Genetically modified cells according to the invention are for example produced by transformation, transduction, conjugation or a combination of these methods with a vector that contains the desired gene, an allele of this gene or parts thereof and a vector that makes expression of the gene possible.
  • Heterologous expression is in particular achieved by integration of the gene or of the alleles in the chromosome of the cell or an extrachromosomally replicating vector.
  • the expression of the aforementioned and all subsequently mentioned enzymes or genes can be detected by means of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration in the gel using appropriate evaluation software. If the increase in enzyme activity is based exclusively on an increase in expression of the corresponding gene, the increase in enzyme activity can be quantified in a simple way by comparing the 1- or 2-dimensional protein separations between wild type and genetically modified cell.
  • a usual method for the preparation of protein gels in the case of coryneform bacteria and for identification of the proteins is the procedure described by Hermann et al. ( Electrophoresis, 22: 1712-23 (2001)).
  • the protein concentration can also be analysed by Western blot hybridization with an antibody that is specific for the protein that is to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989) followed by optical evaluation with appropriate software for determination of concentration (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647).
  • the activity of DNA-binding proteins can be measured by DNA-Band-Shift-Assays (also called gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155).
  • DNA-binding proteins on the expression of other genes can be detected by various well-described methods of reporter gene assay (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989). Intracellular enzymatic activities can be determined by various methods that have been described (Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823).
  • the increase in enzyme activity as well as the decrease in enzyme activity are preferably determined by the methods described in Hermann et al., Electophoresis, 22: 1712-23 (2001), Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001).
  • mutations can either be produced undirected according to classical methods, such as by UV-irradiation or by mutation-causing chemicals, or purposefully by genetic engineering methods such as deletion(s), insertion(s) and/or nucleotide exchange(s). Genetically modified cells are obtained as a result of these mutations.
  • Especially preferred mutants of enzymes are in particular also enzymes for which feedback inhibition is no longer present or at least is reduced in comparison with the wild-type enzyme.
  • the increase in enzyme activity is brought about through an increase in expression of an enzyme, then for example we increase the copy number of the corresponding genes or mutate the promoter and regulating region or the ribosome binding site, which is located upstream of the structural gene.
  • Expression cassettes that are inserted upstream of the structural gene work in this way.
  • the enzyme gene can also be assigned, as regulatory sequences, so-called “enhancers”, which as a result of improved interaction between RNA-polymerase and DNA also bring about increased gene expression. Expression is also improved by measures for extending the life of the m-RNA.
  • enzyme activity is also intensified.
  • genes or gene constructs are then either contained in plasmids with varying copy number or are integrated in the chromosome and amplified. Alternatively, overexpression of the relevant genes can in addition be achieved by altering the composition of the medium and the culture conditions.
  • a person skilled in the art will find instructions for this in, inter alia, Martin et al. ( Bio/technology 5, 137-146 (1987)), Guerrero et al. ( Gene 138, 35-41 (1994)), Tsuchiya and Morinaga ( Bio/technology 6, 428-430 (1988)), Eikmanns et al. ( Gene 102, 93-98 (1991)), in EP-A-0 472 869, in U.S. Pat. No.
  • Episomal plasmids are used for increasing the expression of the genes in question. Suitable plasmids are in particular those that are replicated in coryneform bacteria. Numerous known plasmid vectors, for example pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKExl (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
  • plasmid vectors for example those based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66: 119-124 (1990)) or pAG1 (U.S. Pat. No. 5,158,891), can be used in the same way.
  • plasmid vectors are also suitable, by means of which we can apply the method of gene amplification by integration into the chromosome, as was described for example by Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for the duplication or amplification of the hom-thrB operon.
  • this method the complete gene is cloned into a plasmid vector, which can be replicated in a host (typically Escherichia coli ), but not in Corynebacterium glutamicum .
  • Vectors that may be considered are for example pSUP301 (Simon et al., Bio/Technology 1: 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145: 69-73 (1994)), pGEM-T (Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994)), pCR® Blunt (Invitrogen, Groningen, The Netherlands), pEM1 (Schrumpf et al., Journal of Bacteriology 173: 4510-4516)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)).
  • the plasmid vector that contains the gene to be amplified is then transferred by conjugation or transformation into the desired strain of Corynebacterium glutamicum .
  • the method of conjugation is described for example in Schwarzerbach et al., Applied and Environmental Microbiology 60: 756-759 (1994). Methods for transformation are described for example in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio/Technology 7: 1067-1070 (1989) and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994).
  • the resultant strain contains at least two copies of the relevant gene.
  • the formulation used in the above and hereinafter “increased activity of an enzyme E x relative to its wild type” preferably always means activity of the respective enzyme that is increased by a factor of at least 2, especially preferably of at least 10, even more preferably of at least 100, yet more preferably of at least 1000 and most preferably of at least 10000.
  • the cell according to the invention which has “increased activity of an enzyme E x relative to its wild type”, in particular also comprises a cell whose wild type has no or at least no detectable activity of this enzyme E x and only displayed a detectable activity of this enzyme E x after the enzyme activity was increased, for example through overexpression.
  • the term “overexpression” or the formulation “increase in expression” used hereinafter also includes the case when a starting cell, for example a wild-type cell, has no or at least no detectable expression and it is only by recombinant methods that a detectable expression of the enzyme E x is induced.
  • the cell it is preferable for the cell to have increased activity of enzymes E I and E II , enzymes E I and E III , enzymes E II and E III or even increased activity of all the enzymes E I , E II and E III .
  • a preferred enzyme E I in particular a preferred alkane monoxygenase is the alkane monoxygenase encoded by the alkBGT gene from Pseudomonas putida GPO1.
  • the isolation of the alkBGT gene sequence is described for example by van Beilen et al. in “ Functional Analysis of Alkane Hydroxylases from Gram - Negative and Gram - Positive Bacteria ”, Journal of Bacteriology, Vol. 184 (6), pages 1733-1742 (2002).
  • cytochrome P450 monoxygenases in particular cytochrome P450 monoxygenases from Candida , for example from Candida tropicalis , or from plants, for example from the chick-pea ( Cicer arietinum L.
  • cytochrome P450 monoxygenases can also be used as alkane monoxygenases.
  • the gene sequences of suitable cytochrome P450 monoxygenases from Candida tropicalis are for example disclosed in WO-A-00/20566, whereas the gene sequences of suitable cytochrome P450 monoxygenases from the chick-pea are given for example by Barz et al. in “ Cloning and characterization of eight cytochrome P 450 cDNAs from chickpea ( Cicer arietinum L. ) cell suspension cultures”, Plant Science, Vol. 155, pages 101-108 (2000).
  • Other homologues of the alkB gene are also given by van Beilen et al. in “ Oil & Gas Science and Technology ”, Vol.
  • a suitable gene for a xylene monooxygenase is for example the xylM or the xylA gene, and a plasmid containing these two genes has the GENBANK Accession No. M37480.
  • a preferred enzyme E II in particular a preferred alcohol dehydrogenase is for example the alcohol dehydrogenase encoded by the alkJ gene (EC 1.1.99-2), in particular the alcohol dehydrogenase encoded by the alkJ gene from Pseudomonas putida GPo1.
  • the gene sequences the alcohol dehydrogenase encoded by the alkJ gene from Pseudomonas putida GPo1, Alcanivorax borkumensis, Bordetella parapertussis, Bordetella bronchiseptica or from Roseobacter denitrificans can be found for example in the KEGG gene databank.
  • Suitable ⁇ -transaminases are for example the ⁇ -transaminases that are characterized in US-A-2007/0092957 by the sequence numbers 248, 250, 252 and 254.
  • a preferred enzyme E III in particular a preferred ⁇ -transaminase is in particular the ⁇ -transaminase from Chromobacterium violaceum DSM30191 (Kaulmann et al., 2007; “ Substrate spectrum of ⁇ - transaminase from Chromobacterium violaceum DSM 30191 and its potential for biocatalysis”, Enzyme and Microbial Technology , Vol. 41, pages 628-637), which is encoded by the gene sequence according to SEQ ID No. 01.
  • ⁇ -transaminases that can be isolated from plants.
  • the ⁇ -transaminases from plants selected from the group comprising Arabidopsis thaliana, Avena saliva, Beta vulgaris, Glycine max, Hordeum vulgare, Lotus japonicus, Solanum lycopersicum, Manihot esculenta, Oryza sativa, Traeticum aestivum, Zea mays, Spinacia oleracea, Arum maculatum, Mercurialis perennis and Urtica dioica , are preferred here, and Arabidopsis thaliana is especially preferred.
  • Enzymes that are encoded by nucleic acids that have 90%, preferably 95%, especially preferably 99 and quite especially preferably 100% identity to the sequence according to SEQ ID No. 39 are suitable in particular as ⁇ -transaminases.
  • the “nucleotide identity” relative to SEQ ID No. 39 is determined using known methods. In general, special computer programs with algorithms are used, taking into account special requirements. Preferred methods for determination of identity first produce the greatest agreement between the sequences to be compared.
  • Computer programs for determination of identity comprise, but are not restricted to, the GCG software package, including
  • the well-known Smith-Waterman algorithm can also be used for determining nucleotide identity.
  • Preferred parameters for nucleotide comparison comprise the following:
  • the GAP program is also suitable for use with the parameters given above.
  • the aforementioned parameters are the default parameters in the nucleotide sequence comparison.
  • enzymes from the subgroup of the ⁇ -Ala:pyruvate transaminases are suitable. These include e.g. transaminases from Pseudomonas putida W619 (gi: 119860707, gi: 119855857, gi: 119856278), from Pseudomonas putida KT2440 (gi: 24984391), from Pseudomonas aeruginosa PA01 (gi 15595330, gi: 15600506, gi 15595418, gi 9951072); Streptomyces coelicolor A3(2) (gi: 3319731), Streptomyces avermitilis MA 4680 (gi: 29831094, gi: 29829154) and Chromobacterium violaceum ATCC 12472 (gi 34102747).
  • the amino acid sequences of the aforementioned transaminases are presented in the
  • the cell according to the invention has, in addition to increased activity of at least one of the enzymes E I , E II and E III , preferably in addition to increased activity of the enzymes E I and E III or E I , E II and E III , also increased activity of an enzyme E IV , which catalyses the conversion of ⁇ -aminocarboxylic acid esters to the corresponding ⁇ -aminocarboxylic acids, said enzyme E IV preferably being an esterase, which preferably is secreted by the cell.
  • Preferred esterases according to the invention are in particular lipase, and as an example of a suitable lipase we may mention the lipase LipA from Pseudomonas fluorescens HU380 (ACC Code Q76D26, Kojima and Shimizu, “ Purification and Characterization of the Lipase from Pseudomonas fluorescens HU 380 ”, Journal of Bioscience and Bioengineering . Volume 96 (3), pages 219-226 (2003)).
  • the esterases are secreted, they can be provided, in a manner known by a person skilled in the art, with corresponding signal sequences, which establish secretion.
  • LipA from Pseudomonas fluorescens HU380 is overexpressed in E. coli , it can be provided advantageously with signal sequences from EstA, an esterase that occurs naturally on the cell surface of Pseudomonas aeruginosa (Becker et al., “ A generic system for the Escherichia coli cell - surface display of lipolytic enzymes”, FEBS Letters , Vol. 579, pages 1177-1182 (2005)).
  • Other suitable enzymes are lipases from C. antarctica, M. miehei and P. cepacia (Vaysse et al., “ Enzyme and Microbial Technology ”, Vol. 31, pages 648-655 (2002)).
  • the secreted ⁇ -aminocarboxylic acid ester can also be cleaved conventionally, to obtain the ⁇ -aminocarboxylic acid, for example by saponification, i.e. hydrolysis of the ⁇ -aminocarboxylic acid ester by the aqueous solution of a hydroxide, e.g. by sodium hydroxide.
  • saponification i.e. hydrolysis of the ⁇ -aminocarboxylic acid ester by the aqueous solution of a hydroxide, e.g. by sodium hydroxide.
  • the cell according to the invention in addition to increased activity of at least one of the enzymes E I , E II and E III , preferably in addition to increased activity of the enzymes E I and E III or E I , E II and E III , and optionally also in addition to increased activity of the aforementioned enzyme E IV , also has increased activity of an enzyme E V , which catalyses the conversion of ⁇ -aminocarboxylic acids to the corresponding lactams, and it can also be advantageous here if this enzyme E V is secreted by the cell.
  • the cell according to the invention has, in addition to increased activity of one or more of the enzymes E I , E II or E III and optionally increased activity of the enzyme E IV and/or E V , also increased activity of an enzyme E VI , which catalyses the conversion of an ⁇ -ketocarboxylic acid to an amino acid, said enzyme E VI preferably being an amino acid dehydrogenase.
  • Such a modification of the cell would have the advantage that in the case when amino acids are used as donor for the NH 2 group, which is consumed during the transaminase (E III )—mediated reaction of an ⁇ -oxocarboxylic acid or an ⁇ -oxocarboxylic acid ester to the corresponding ⁇ -aminocarboxylic acid, to the corresponding ⁇ -aminocarboxylic acid ester or to the corresponding ⁇ -aminocarboxylic acid ester, can be correspondingly regenerated.
  • amino acid dehydrogenase is the alanine dehydrogenase from B. subtilis (EC No.
  • Suitable amino acid dehydrogenases are serine dehydrogenases, aspartate dehydrogenases, phenylalanine dehydrogenases and glutamate dehydrogenases.
  • a contribution to achievement of the aims stated at the beginning is also provided by a method for the production of a genetically modified cell, comprising the process step of increasing the activity of at least one of the following enzymes:
  • the activity of an enzyme E IV which catalyses the conversion of ⁇ -aminocarboxylic acid esters to the corresponding ⁇ -aminocarboxylic acids
  • an enzyme E V which catalyses the conversion of ⁇ -aminocarboxylic acids to the corresponding lactams
  • step I) of the method according to the invention the cells are first brought into contact with a culture medium containing a carboxylic acid or a carboxylic acid ester or with a culture medium contiguous with an organic phase containing a carboxylic acid or a carboxylic acid ester, and this contacting takes place under conditions that make it possible for the cell to form ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids, from the carboxylic acid or from the carboxylic acid esters.
  • the genetically modified cells according to the invention can be brought into contact with the nutrient medium, and therefore cultivated continuously or discontinuously in a batch process or in a fed-hatch process or in a repeated-fed-batch process, for the purpose of producing ⁇ -aminocarboxylic acids or lactams derived from ⁇ -aminocarboxylic acids.
  • a semi-continuous process is also conceivable, as described in GB-A-1009370.
  • Known culture methods are described in Chmiel's textbook (“ Bioreatechnik 1. Consumable Biovontechnik ” [Bioprocess Techniques 1.
  • the culture medium to be used must be suitable for the requirements of the particular strains. Descriptions of culture media for various microorganisms are given in “ Manual of Methods for General Bacteriology ” of the American Society for Bacteriology (Washington D.C., USA, 1981).
  • the carbon source used can be carbohydrates, e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, e.g. soya oil, sunflower oil, peanut oil and coconut oil, fatty acids e.g. palmitic acid, stearic acid and linoleic acid, alcohols e.g. glycerol and methanol, hydrocarbons such as methane, amino acids such as L-glutamate or L-valine or organic acids e.g. acetic acid. These substances can be used separately or as a mixture.
  • carbohydrates e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose
  • oils and fats e.g. soya oil, sunflower oil, peanut oil and coconut oil, fatty acids e.g. palmitic acid, stearic acid and linoleic acid, alcohols e
  • carbohydrates especially monosaccharides, oligosaccharides or polysaccharides, is especially preferred, as described in U.S. Pat. Nos. 6,013,494 and 6,136,576, and of C 5 -sugars or glycerol.
  • Organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean flour and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used as the nitrogen source.
  • the nitrogen sources can be used separately or as a mixture.
  • Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus.
  • the culture medium must in addition contain salts of metals, for example magnesium sulphate or iron sulphate, which are required for growth.
  • essential growth substances such as amino acids and vitamins are used in addition to the substances mentioned above.
  • suitable precursors can be added to the culture medium. The stated substances can be added to the culture in the form of a single preparation, or they can be supplied in a suitable manner during cultivation.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulphuric acid are used in a suitable manner for controlling the pH of the culture.
  • Antifoaming agents e.g. fatty acid polyglycol esters, are used for controlling foaming.
  • suitable selectively acting substances e.g. antibiotics, can be added to the medium.
  • oxygen or oxygen-containing gas mixtures e.g. air, are fed into the culture.
  • the temperature of the culture is normally in the range from 20° C. to 45° C. and preferably 25° C. to 40° C.
  • a recombinant cell derived from an E. coli cell
  • a mineral salt medium supplemented with ampicillin, chloramphenicol and kanamycin according to Riesenberg et al., “ High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1”, Appl Microbiol and Biotechnololgy , Vol. 34 (1), pages 77-82 (1990)
  • a mineral salt medium supplemented with ampicillin, chloramphenicol and kanamycin according to Riesenberg et al., “ High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1”, Appl Microbiol and Biotechnololgy , Vol. 34 (1), pages 77-82 (1990)
  • the nutrient medium is used as the nutrient medium.
  • the contacting of the cells according to the invention with the culture medium in step I) preferably takes place in conditions that enable the cell to form ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids starting from carboxylic acid or from carboxylic acid esters.
  • carboxylic acids or carboxylic acid esters consideration may be given in particular to carboxylic acids with number of carbons in the range from 6 to 20, especially preferably from 6 to 15, in particular from 6 to 12, lauric acid being especially preferred as carboxylic acid.
  • carboxylic acid esters consideration may be given in particular to the methyl or ethyl esters of the aforementioned carboxylic acids, with the methyl ester of lauric acid being especially preferred as carboxylic acid ester.
  • step I In the production of the ⁇ -aminocarboxylic acids, ⁇ -aminocarboxylic acid esters or lactams derived from ⁇ -aminocarboxylic acids, various procedures are conceivable in step I).
  • the cells are first cultivated, for the purpose of biomass production, in a nutrient medium that does not contain carboxylic acids or carboxylic acid esters, and in particular does not contain the aforementioned, preferred carboxylic acids or carboxylic acid esters. It is only after a certain biomass has been obtained that the carboxylic acids or the carboxylic acid esters are added to the nutrient medium or the cells are brought into contact with a new nutrient medium containing the carboxylic acids or carboxylic acid esters.
  • the content of carboxylic acids or carboxylic acid esters during the formation of ⁇ -aminocarboxylic acids, of ⁇ -aminocarboxylic acid esters or of lactams derived from ⁇ -aminocarboxylic acids prefferably in the range from 1 to 200 g/l, especially preferably in the range from 20 to 200 g/l.
  • the cells first to be cultivated, for the purpose of biomass production, in a nutrient medium that does not contain carboxylic acids or carboxylic acid esters, and in particular does not contain the aforementioned, preferred carboxylic acids or carboxylic acid esters. It is only after a certain biomass has been obtained that the cell suspension as aqueous phase A) is brought into contact with the organic phase B), where in particular the organic phase B) contains the carboxylic acid or the carboxylic acid esters preferably in an amount in the range from 1 to 200 g/l, especially preferably in the range from 20 to 200 g/l.
  • carboxylic acids or carboxylic acid esters are employed as carboxylic acids or carboxylic acid esters, then the content of these carboxylic acids or carboxylic acid esters in the organic phase can also be significantly higher. In such a case it may also be possible to use the pure carboxylic acid or the pure carboxylic acid esters, for example pure methyl laurate, as organic phase.
  • alkanes of medium chain length preferably those with a logP value of more than 4 (little foam formation), or physically similar aromatics or aromatic esters, though preferably, as mentioned above, lauric acid esters, especially preferably methyl laurate, BEHP (bis(2-ethylhexyl)phthalate) or long-chain fatty acid esters (biodiesel).
  • lauric acid esters especially preferably methyl laurate, BEHP (bis(2-ethylhexyl)phthalate) or long-chain fatty acid esters (biodiesel).
  • the culture medium used in step I) contains amino group donors, such as ammonia or ammonium ions or even amino acids, though in particular alanine or aspartate, which function as amine donors in the transaminase-catalysed conversion of the ⁇ -oxocarboxylic acids or the ⁇ -oxocarboxylic acid esters to the corresponding ⁇ -aminocarboxylic acids or ⁇ -aminocarboxylic acid esters.
  • amino group donors such as ammonia or ammonium ions or even amino acids, though in particular alanine or aspartate, which function as amine donors in the transaminase-catalysed conversion of the ⁇ -oxocarboxylic acids or the ⁇ -oxocarboxylic acid esters to the corresponding ⁇ -aminocarboxylic acids or ⁇ -aminocarboxylic acid esters.
  • step II) of the method according to the invention the resultant ⁇ -aminocarboxylic acids, the resultant ⁇ -aminocarboxylic acid esters or the lactams derived from the ⁇ -aminocarboxylic acids are optionally isolated, and it is preferable for this isolation to take place in an at least two-stage purification process, comprising
  • the extraction in step a) can in particular be designed as so-called “in situ” extraction, in which steps I) and II) of the method according to the invention for the production of ⁇ -aminocarboxylic acids, of ⁇ -aminocarboxylic acid esters or of lactams derived from ⁇ -aminocarboxylic acids are carried out simultaneously.
  • This “in situ” extraction has already been described above.
  • the fine purification in step II) can for example take place by distillation or crystallization.
  • the ⁇ -aminocarboxylic acid esters formed in step I) are reacted in another process step by conventional chemical methods to ⁇ -aminocarboxylic acids;
  • a preferred, conventional chemical method is saponification, in which the ⁇ -aminocarboxylic acid ester is reacted with an aqueous solution of a base, preferably a hydroxide, preferably sodium hydroxide, to the ⁇ -aminocarboxylic acid.
  • this method is used for the production of ⁇ -aminolauric acid from lauric acid esters, preferably methyl laurate;
  • ⁇ -aminocarboxylic acids ⁇ -aminocarboxylic acid esters or by lactams derived from ⁇ -aminocarboxylic acids, which are obtainable by the method described above, the lactam preferably being laurinlactam, which is obtained if lauric acid or lauric acid esters are used as carboxylic acid or as carboxylic acid esters in step I) of the method according to the invention for the production of lactams derived from ⁇ -aminocarboxylic acids, wherein the ⁇ -aminocarboxylic acid is preferably ⁇ -aminolauric acid and the ⁇ -aminocarboxylic acid ester is preferably ⁇ -aminomethyl laurate.
  • step ( ⁇ 2) of the method according to the invention for the production of polyamides based on ⁇ -aminocarboxylic acids the ⁇ -aminocarboxylic acids obtained in step ( ⁇ 1), in particular the ⁇ -aminolauric acids obtained in step ( ⁇ 1), are converted in a polymerization to a polyamide, and optionally mixtures of various ⁇ -aminocarboxylic acids can also be used, for which at least one of the ⁇ -aminocarboxylic acids, but optionally all ⁇ -aminocarboxylic acids were produced by the method according to the invention for the production of ⁇ -aminocarboxylic acids.
  • the production of the polyamides from the ⁇ -aminocarboxylic acids can be carried out by well-known methods, as described for example in L. Notarbartolo, Ind. Plast. Mod. 10 (1958)2, p. 44, JP 01-074224, JP 01-051433, JP63286428, JP58080324 or JP60179425.
  • step ( ⁇ 2) of the method according to the invention for the production of polyamides based on lactams the lactams obtained in step ( ⁇ 1), in particular the laurinlactam obtained in step ( ⁇ 1), are converted in a ring opening polymerization or by polycondensation to a polyamide, and optionally it is also possible to use mixtures of various lactams, for example mixtures of laurinlactam and ⁇ -caprolactam, for which at least one of the lactams, but optionally all lactams were produced by the method according to the invention for the production of lactams derived from ⁇ -aminocarboxylic acids.
  • the production of the polyamides from the lactams can be carried out by well-known methods, as described for example in DE-A-14 95 198, DE-A-25 58 480, EP-A-0 129 196 or also in “ Polymerization Processes ”, Interscience, New York, 1977, pages 424-467, especially pages 444-446.
  • polyamides which are obtainable by the methods described above. It is especially preferable for these polyamides to be based, up to at least 10 wt. %, especially preferably up to at least 25 wt. %, still more preferably up to at least 50 wt. % and most preferably up to at least 75 wt. %, on lauric acid, lauric acid ester or laurinlactam, obtained by the method according to the invention for the production of lauric acid, of lauric acid ester or of laurinlactam from lauric acid or lauric acid esters.
  • FIG. 1 shows, schematically, a recombinant plasmid for chromosomal integration of the alk genes in Escherichia coli (tnp: transposase gene; bla: ampicillin resistance gene; oriT RP4: mobilization region; I and O mark the “inverted repeats” of the mini-transposon Tn5).
  • FIG. 2 shows, schematically, a recombinant plasmid for expression of transaminase and of alanine dehydrogenase under the control of an arabinose inducible promoter (bla: ampicillin resistance gene; CV2025: gene for ⁇ -transaminase from Chromobacterium violaceum ; ald: gene for alanine dehydrogenase from B. subtilis ; araC: transcription regulator).
  • bla ampicillin resistance gene
  • CV2025 gene for ⁇ -transaminase from Chromobacterium violaceum
  • ald gene for alanine dehydrogenase from B. subtilis
  • araC transcription regulator
  • FIG. 3 shows, schematically, a recombinant plasmid for the expression of LipA in E. coli and presentation of the enzyme on the cell surface (colE1, ColE1: replication origin; estA*, estA: gene with amino acid exchange alanine for serine (codon #38), cat: chloramphenicol resistance gene; phoA: gene segment that encodes the leader sequence of alkaline phosphatase).
  • FIG. 4 shows determination of the activity of C. violaceum -transaminase from the enzyme assay. The activity was determined in duplicate (active1, active2) with a photometer. A batch without the ⁇ -substrate L-alanine (w.Cos), a batch with heat-inactivated enzyme (inactive) and a batch from E. coli expression culture with empty vector (empty vector), purified similarly to the omega-TA, were used as negative controls.
  • active1, active2 ⁇ -substrate L-alanine
  • inactive heat-inactivated enzyme
  • empty vector empty vector
  • FIG. 5 shows chromatograms of the substrate 12-aminomethyl laurate at the start of reference measurement (top) and after 2 h incubation with the purified transaminase (bottom).
  • FIG. 6 shows chromatograms of the substrate 12-aminomethyl laurate after 24 h (top) of the enzyme assay and after spiking the substrate (control, bottom).
  • FIG. 7 shows 6 chromatograms of the substrate 12-aminomethyl laurate after heat inactivation of the enzyme (top) and after Oh (bottom).
  • FIG. 8 shows the starting plasmid pGEc47, which was used as template for the amplification of alkBGTS.
  • FIG. 9 shows the primers (SEQ ID NOS: 32-35, respectively, in order of appearance) used and the resultant PCR products alkBFG and alkT.
  • FIG. 10 shows the recombinant vector pBT10, which was used for the synthesis of hydroxymethyl laurate and oxomethyl laurate.
  • FIG. 11 shows a GC chromatogram of the 12-oxo-methyl laurate standard.
  • FIG. 12 shows a GC chromatogram of the 12-hydroxymethyl laurate standard.
  • FIG. 13 shows a chromatogram of the organic phase from a resting cell biotransformation in the bioreactor of methyl laurate, at time 0 min.
  • FIG. 14 shows a chromatogram of the organic phase from a resting cell biotransformation in the bioreactor of methyl laurate, at time 150 min.
  • FIG. 15 shows the expression vector pGJ3130 with AT3G22200.
  • FIG. 16 shows detection of enzyme activity by coupled enzyme assay (inactive, heat-inactivated protein; w.Cos., without addition of alanine; akt, the purified, active enzyme).
  • FIG. 17 shows detection of the heterologously expressed protein AT3G22200 by HPLC. Top: product after 20 min incubation at 10.8 min; bottom: reference substance at 10.8 min.
  • FIG. 18 shows the plasmid map of the expression vector pET-21a(+) with the transaminase gene ppta5 (pPPTA5).
  • FIG. 19 shows the plasmid map of the expression vectors pET-21a(+) with the transaminase gene psta (pPSTA).
  • FIG. 20 shows the amination of 5 mM 12-oxomethyl laurate with the transaminases PPTA5.
  • the peak areas of 12-oxo- and 12-aminomethyl laurate from the chromatograms obtained from neutral and acid extraction were added together and the percentage of educt or product was calculated.
  • FIG. 21 shows the amination of 5 mM 12-oxomethyl laurate with the transaminases PSTA.
  • the peak areas of 12-oxo- and 12-aminomethyl laurate from the chromatograms obtained from neutral and acid extraction were added together and the percentage of educt or product was calculated.
  • E. coli For the conversion of lauric acid or of methyl laurate to laurinlactam, E. coli is supplemented with the necessary enzymes monooxygenase, alcohol dehydrogenase, ⁇ -transaminase, alanine dehydrogenase and a lipase.
  • the enzymes are overexpressed in E. coli ; the expression levels of the individual enzymes are dependent on the kinetics of the individual reactions and require optimum adjustment to one another. The expression level is adjusted by adding the appropriate amount of inductor.
  • the alkane hydroxylase system alkBGT from Pseudomonas putida GPo1 is used for the hydroxylation of lauric acid or of methyl laurate.
  • the second step to the aldehyde is catalysed by the alcohol dehydrogenase alkJ.
  • the genes alkBGJT necessary for these reactions are integrated in E. coli by insertion into the mini-transposon Tn5 chromosomally into the genome of E. coli (de Lorenzo et al., J. Bacteriol ., Vol. 172 (11), pages 6568-6572 and 6557-6567 (1990); Panke et al., Appl and Environm. Microbiol ., pages 5619-5623 (1999)).
  • the genes are to be expressed under the control of the alkB promoter and of the positive regulator alkS. Transfer of the Tn5-alkBGFJST construct to the E. coli target organism is effected with the aid of the mobilizable plasmid pUT-Km (Panke et al., 1999, see above).
  • the alkST locus with the expression-relevant regulator alkS is organized outside of the alkBFGHJKL operon and arranged in the opposite direction in the genome of Pseudomonas putida .
  • the arrangement of the genes is preserved during cloning into the transposon-bearing plasmid.
  • the fragments alkST and alkBFGJ are integrated into the plasmid one after another.
  • alkB SEQ ID No. 03
  • alkG SEQ ID No. 04
  • alkJ SEQ ID No. 05
  • alkB and alkG To simplify the cloning of alkB and alkG, the gene alkF located between them is amplified and cloned together with alkB and alkG. A1kF is of no significance for the reaction that is to be catalysed.
  • the genes alkBFG and alkJ are amplified in two separate PCR steps and fused together by SOE-PCR (Horton et al., Gene , Vol. 77, pages 61-68 (1989)).
  • the OCT plasmid from Pseudomonas putida GPo1 serves as target DNA.
  • the PCR fragment is cloned into the transposon-bearing vector pUT-Km.
  • the vector is cut within Tn5 with NotI and ligated with the alkST fragment by blunt end ligation.
  • the recombinant plasmid is designated pUT-Km-alkST.
  • the two separate fragments are fused together by SOE-PCR. (3 separate PCR reactions required).
  • the recombinant plasmid pUT-Km-alkST and the alkBFGJ construct are cut with NotI and ligated.
  • the new recombinant plasmid pUT-Km-alkBGJST (see FIG. 1 ) is transformed in E. coli HB101 and transferred to E. coli JM101 by conjugative plasmid transfer.
  • the Tn5:alkBGJST-bearing E. coli strain JM101 is additionally transformed with the recombinant plasmid pBAD-CV2025-aid.
  • This plasmid is based on the pBAD30 vector (Guzman et al., “ Tight Regulation, Modulation, and High - Level Expression by Vectors Containing the Arabinose P BAD Promoter”, J. Bacteriol , Vol. 177 (14), pages 4121-4130 (1995)).
  • pBAD-CV2025-ald carries the gene for the transaminase CV2025 from Chromobacterium violaceum DSM30191 (SEQ ID No. 01; Kaulmann et al., Enzyme and Microbial Technology Vol. 41, pages 628-637 (2007) and the gene ald, which codes for an alanine dehydrogenase from Bacillus subtilis subsp. Subtilis (SEQ ID No. 02; NP — 391071). The genes are under the control of an arabinose inducible promoter.
  • the forward-primer contains a ribosome binding site in addition to the XbaI cleavage site. Ligation into the pBAD30 vector takes place via the cleavage sites SacI and KpnI.
  • the recombinant vector is designated pBAD-CV2025.
  • the forward-primer contains a ribosome binding site in addition to the XbaI cleavage site.
  • the cloning into the vector takes place via the cleavage sites XbaI and PstI.
  • the resultant plasmid is designated pBAD-CV2025-ald (see FIG. 2 ).
  • the gene coding for omega-transaminase was synthesized by the company Geneart, optimized for E. coli codon usage (SEQ ID No. 42) and cloned into the vector pGA15 (Geneart). During synthesis of the gene, the cleavage sites SacI and KpnI were incorporated in flanking positions and, after digestion with Sad and KpnI, were cloned into the vector pGA15, linearized beforehand with Sad and KpnI. The resultant vector was digested with the restriction endonucleases SacI and KpnI, the fragment (transaminase) was purified and was ligated into the expression vector paCYCDuet-1 (Novagen). The correct plasmids were verified by restriction analysis. The resultant vector is called paCYCDuet-1::omega tranaminase.
  • Lysis of the bacterial expression culture 50 ml of culture was centrifuged at 2360 x g, and then resuspended in 5 ml Na-phosphate buffer (pH 8) with 5 mM EDTA, 300 mM NaCl and 1 mg/ml lysozyme, and incubated for 1 h at RT. The lysate was centrifuged at 2360 x g for 10 min and the supernatant was purified on a Protino Ni-TED 2000 packed column (following the instructions of the manufacturer; Macherey-Nagel, Düren). The protein concentration was determined according to Bradford.
  • the activity was determined in a coupled assay, in which the pyruvate formed as by-product of the transaminase reaction is reacted further in a second step, and NADH is oxidized to NAD+.
  • the decrease in NADH concentration (principle: measurement of the decrease in extinction) is measured in the photometer at 340 nm and provides a measure of the activity.
  • the assay was started by adding 5 ⁇ l 12-ODME (50 mg/ml). Measurement is performed continuously every minute at 340 nm at RT up to max. 20 minutes.
  • Inactivated protein and a preparation without ⁇ -substrate were used for control.
  • FIG. 4 shows the variation in extinction, determined photometrically.
  • Solvent B was acetonitrile with 5% 50 mM NaAC pH 4. The gradient was from 30% B to 60% B in 4 min, from 60% B to 100% B in 2 min. The flow rate was 1.2 ml/min. Separation took place in an Agilent Zorbax RP18 column (Agilent Technologies, USA), the column temperature was 40° C.
  • FIGS. 5 and 6 show the standard and the decrease of the 12-oxomethyl laurate. Heat-inactivated enzyme was used as negative control ( FIG. 7 ).
  • the lipase LipA (Q76D26) from Pseudomonas fluorescens (Kojima & Shimizu, J. of Bioscience and Bioengin ., Vol. 96 (3), pages 219-226 (2003)) is used for the cleavage of ⁇ -aminomethyl laurate to ⁇ -aminolauric acid.
  • the gene is amplified with the primers LipA-SfiI-up and LipA-SfiI-down with chromosomal DNA from Pseudomonas fluorescens and cloned via the SfiI cleavage sites into the vector pEST100.
  • the recombinant plasmid is designated pEST-lipA (see FIG. 3 ).
  • the cloning fuses lipA (SEQ ID No. 18) to the signal sequence of alkaline phosphatase phoA and the autotransporter domain of EstA, an esterase from P. aeruginosa , so that the lipase is transferred across the cytoplasmic membrane and is displayed on the cell surface of E. coli .
  • lipA SEQ ID No. 18
  • EstA an esterase from P. aeruginosa
  • the construct pBT10 ( FIG. 10 , SEQ ID No. 31), which contains the three components alkane hydroxylase (AlkB), rubredoxin (AlkG) and rubredoxin reductase (AlkT) from Pseudomonas putida that are necessary for the oxidation to the aldehyde, was produced starting from the pCOM systems (Smits et al., 2001 Plasmid 64:16-24).
  • the alkBFG gene sequence was put under the control of the alkB-promoter and the alkT gene under the control of the alkS-promoter.
  • alkF located between them was amplified and cloned together with alkB and alkG.
  • AlkF is of no significance for the reaction that is to be catalysed.
  • alkBFG forward (SEQ ID No. 32) AAGGGAATTCCATATGCTTGAGAAACACAGAGTTC
  • alkBFG reverse (SEQ ID No. 33) AAAATTCGCGTCGACAAGCGCTGAATGGGTATCGG
  • Primer alkT forward (SEQ ID No. 34) TGAGACAGTGGCTGTTAGAG
  • Primer alkT reverse (SEQ ID No. 35) TAATAACCGCTCGAGAACGCTTACCGCCAACACAG
  • the fragments alkBFG and alkT were amplified by PCR.
  • the plasmid pGEc47 ( FIG. 12 ) (Eggink et al. (1987) J. Biol. Chem. 262, 17712-17718) was used as template.
  • the clonings were carried out by means of the subcloning vector pSMART® HCKan (Lucigen Corporation). This additional step was necessary because direct cloning had not been successful.
  • the commercially available vector pSMART® HCKan (Lucigen) which was already linearized and provided with blunt ends, was ligated with the respective blunt-end PCR product ( FIG. 9 ).
  • the alkBFG fragment with the restriction enzymes NdeI and SalI and the alkT fragment with the restriction enzymes PacI and XhoI were cut out of the subcloning vectors.
  • the fragments were separated in agarose gel (1%), cut out of the gel and isolated using a gel extraction kit.
  • the fragments were ligated one after another into the vector pCOM10 (Smits, T. H. M., Seeger, M. A., Witholt, B. & van Beilen, J. B. (2001) Plasmid 46, 16-24).
  • alkBFG was inserted in pCOM10 via the cleavage sites NdeI and SalI, and in a second step alkT was then cloned via the cleavage sites PacI and XhoI.
  • the recombinant plasmid was first transformed in E. coli DH5 ⁇ . Plasmid-bearing clones were selected on kanamycin-containing LB medium. The isolated plasmid was checked by restriction analysis and sequencing. It is designated pBT10 ( FIG. 10 ).
  • the plasmid pBT10 was transformed by heat shock at 42° C. for 2 min in the chemically competent strain E. coli W3110.
  • the cells were centrifuged, the cell pellet was resuspended in KPi-buffer (50 mM, pH 7.4) and put in a bioreactor. A biomass concentration of about 1.8 gCDW/L was established. Stirring vigorously (1500 min ⁇ 1 ), the substrate methyl laurate in the ratio 1:3 was added to the cell suspension (100 ml cell suspension, 50 ml methyl laurate). The temperature was kept constant at 30° C. Formation of the products hydroxymethyl laurate and 12-oxomethyl laurate was detected by GC analysis of the reaction mixture. For this, a sample was taken after 0 min as negative control ( FIG. 13 ) and after 150 min ( FIG.
  • a known aminotransferase from Arabidopsis thaliana was analysed.
  • 4-aminobutyrate transaminase (at3g22200, SEQ ID No. 38) displayed an activity of about 14 U/mg heterologously expressed protein versus 12-oxomethyl dodecanoate.
  • the product 12-aminomethyl dodecanoate was confirmed by HPLC.
  • RNA was isolated with the RNeasy Mini Kit from a whole, flowering plant of the species A. thaliana following the instructions of the manufacturer: QIAGEN GmbH, Hilden.
  • RNA quality/quantity determination was performed by Nanodrop following the instructions of the manufacturer (Thermo Fisher Scientific Inc. Waltham USA).
  • Forward-primer inserts protease cleavage site and NaeI, SEQ ID No. 36 GCCGGCGAGAACCTGTACTTTCAGATGGCAAGTAAGTATGCCACTTG Reverse-primer inserts BamHI, SEQ ID No. 37 GGATCCTCACTTCTTGTGCTGAGCCTTG
  • the resultant PCR product was purified with the NucleoSpin® Extract II Kit (Macherey-Nagel, Germany, following the manufacturer's instructions).
  • the vector pGJ3130 ( FIG. 15 , SEQ ID No. 43) was produced by standard methods of molecular biology and transformed in E. coli XL1blue.
  • Lysis of the bacterial expression culture 50 ml culture was centrifuged at 2360 x g, and then resuspended in 5 ml Na-phophate buffer (pH 8) with 5 mM EDTA, 300 mM NaCl and 1 mg/ml lysozyme, and incubated for 1 h at RT. The lysate was centrifuged at 2360 x g for 10 min and the supernatant was purified on a Protino Ni-TED 2000 packed column (following the instructions of the manufacturer; Macherey-Nagel, Düren). The protein concentration was determined according to Bradford.
  • the activity was determined in a coupled assay, in which the pyruvate that formed as by-product of the transaminase reaction is reacted further in a second step, and NADH is oxidized to NAD+.
  • the decrease in NADH concentration (principle: measurement of the decrease in extinction) is measured in the photometer at 340 nm and provides a measure of the activity.
  • the assay was started by adding 5 ⁇ l 12-ODME (50 mg/ml). Measurement is performed continuously every minute at 340 nm at RT for up to max. 20 minutes.
  • Inactivated protein and a preparation without ⁇ -substrate were used as the control.
  • FIG. 16 shows the variation in extinction, determined photometrically.
  • the preparation was derivatized with o-phthalic aldehyde (oPA) and 250 ⁇ l was analysed.
  • 50 mM NaAC pH 4:acetonitrile 4:1 (v:v) was used as solvent A.
  • Solvent B was acetonitrile with 5% 50 mM NaAC pH 4. The gradient was from 30% B to 60% B in 4 min, from 60% B to 100% B in 2 min. The flow rate was 1.2 ml/min.
  • FIG. 17 shows the formation of 12-aminomethyl laurate.
  • the reference sample is shown at the bottom in FIG. 17 .
  • the strains E. coli BL21(DE3)/PPTA5 and E. coli BL21(DE3)/PSTA were used for the amination of 12-oxomethyl laurate. These strains were constructed as follows.
  • the expression vector pET-21a(+) (Novagen) was selected for the cloning of both transaminase genes.
  • SEQ ID No. 40 primers were constructed, which were intended to add the restriction cleavage sites NdeI and XhoI to the ends of the gene; primer PPTA5_NdeI: GGAATTCCATATGAGCGTCAACAACCCGCAAACCCG (SEQ ID No.
  • Primer PPTA5_XhoI CCGCTCGAGTTATCGAATCGCCTCAAGGGTCAGGTCC (SEQ ID No. 45).
  • SEQ ID No. 41 primers with NdeI and BamHI at the ends; primer PSTA_NdeI: GGAATTCCATATGAGCGATTCGCAAACCCTGCACTGGC (SEQ ID No. 46) and Primer PSTA 13 BamHI: CGCGGATCCTCAGCCCAGCACATCCTTGGCTGTCG (SEQ ID No. 47)
  • the purified PCR products and the vector pET-21a(+) were then submitted to restriction with the restriction enzymes NdeI and XhoI or NdeI and BamHI.
  • the cut vector was dephosphorylated with alkaline phosphatase from shrimp.
  • the vector cut with NdeI and XhoI and the PPTA5 gene, and the vector cut with NdeI and BamHI and the psta gene were, after ligation with T4 DNA ligase, transformed with the competent expression strain E. coli XL1-Blue. After some clones had been grown, the plasmids were isolated and then underwent restriction and gel electrophoretic analysis. The transaminase sequences of the clones obtained (pPPTA5 or pPSTA) were confirmed by sequence analysis.
  • FIG. 18 shows the plasmid maps of the expression vectors.
  • the vectors pPPTA5 and pPSTA were transformed in competent E. coli BL21(DE3) cells.
  • One individual colony of each was inoculated in 5 ml LB-Amp medium (ampicillin concentration 100 ⁇ g/ml) and shaken overnight at 37° C.
  • 1% was inoculated in 200 ml LB-Amp medium, shaken at 37° C. and after reaching an OD 600 of 0.5, gene expression was induced with 0.5 mM IPTG. After shaking for 20 hours at 30° C., the cells were harvested and stored at ⁇ 20° C.
  • the 400 ⁇ l preparations contained 5 mM 12-oxomethyl laurate, dissolved in N,N-dimethylformamide, 500 mM DL-alanine, 1 mM pyridoxal-5′-phosphate and 80 ⁇ l raw extract in 10 mM Kpi-buffer pH 7.0. It was shaken at 25° C. After specified times, 20 ⁇ l samples were taken from each, one portion was made alkaline with 1 ⁇ l 1% NaOH solution and was shaken out with 100 ⁇ l ethyl acetate. The organic phases were investigated by gas chromatography (gas chromatograph from Perkin Elmer, Clarus 500 with flame ionization detector). For this, an Optima 5-column (0.25 ⁇ m, 30 m, 0.25 mm, Macherey-Nagel) was used with programme:
US12/742,318 2007-12-17 2008-12-12 ω-Aminocarboxylic acids, ω-aminocarboxylic acid esters, or recombinant cells which produce lactams thereof Active 2029-01-16 US9012227B2 (en)

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DE102007060705A DE102007060705A1 (de) 2007-12-17 2007-12-17 ω-Aminocarbonsäuren oder ihre Lactame, herstellende, rekombinante Zellen
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PCT/EP2008/067447 WO2009077461A1 (de) 2007-12-17 2008-12-12 ω-AMINOCARBONSÄUREN, ω-AMINOCARBONSÄUREESTER ODER IHRE LACTAME HERSTELLENDE, REKOMBINANTE ZELLEN

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